The experiments in this proposal are designed to increase understanding of the mechanisms by which DNA is repaired in eukaryotic cells. The specific goals are concerned with DNA mismatch repair, the process by which non- complementary bases in the DNA helix are corrected. This pathway in prokaryotes is known to be important in correcting replication errors, deaminations of 5-methylcytosine, and heteroduplexes in DNA which have arisen by homologous recombination. In E. coli, one of the essential enzymes in the mismatch repair pathway is also necessary for repair of UV induced damage, and mismatch repair appears to be involved in repairing potential mutations created by the UV-induced repair pathway. There are also indications that mismatch repair in prokaryotes may play a role in repairing abnormal or damaged DNA that arises due to base analog incorporation, or damage or change by some alkylating agents and mutagens. While defects in DNA repair can clearly lead to various disease states in humans, it remains to be seen whether any diseases are due specifically to defects in the DNA mismatch repair pathway. Increased spontaneous mutation rates could give rise to increased incidences of tumors and other diseases and failure of the pathway could lead to a greater susceptibility to the deleterious effects of radiation, mutagens, and other damaging agents. Compared to the wealth of information known about mismatch repair in prokaryotes, very little is known about mismatch repair in eukaryotes. A mouse gene, Rep-1, has been cloned which likely encodes a central enzyme in the mismatch repair pathway and a partial clone of a homologous gene has also been obtained from yeast. The specific aims of the proposal are: 1) To show that Rep-1 is involved in DNA mismatch repair by measuring spontaneous mutation rates in rep-1 deleted cells, by measuring binding of Rep-1 to mismatched bases, and by testing the importance of Rep-1 in in vitro DNA mismatch repair assays. 2) To determine the various protein products made by the mouse rep-1 gene and what functional differences there might be among the Rep-1 proteins. 3) To clone and characterize the yeast rep-1 homologous gene and determine the role of its gene products in DNA mismatch repair.